It has been suggested recently that hCG has intrinsic thyrotropic activity and infact is the thyroid stimulator of molar pregnancy. Our own extensive in vitro studies, however, have failed to unearth any evidence to support this conclusion. We proposed to seek evidence of and, if present, to isolate any thyroid stimulators from urine of patients with hydatidiform mole, and from the molar tissue itself. The urine will be fractionated by ammonium sulfate and the active fraction purified by ion-exchange and gel permeation chromatographies. Techniques such as isoelectric focusing and affinity chromatography will also be employed, whenever appropriate. Thyrotropic and gonadotropic activities will be monitored by in vitro and in vivo assays. In preliminary studies, we have isolated a potent hCG fraction from molar pregnancy urine that is devoid of TSH activity, and a fraction that TSH activity but has little or no hCG activity in in vitro assays. The purification of the thyroid stimulators will be followed by studies of their physicochemical properties. If the thyroid stimulators are found to be composed of subunits, attempts will be made to dissociate them and to produce hybrid molecules by combination of complementary subunits from different hormones. Such recombinant molecules will be studied for their physiological properties. If any of the purified stimulators become available, an attempt will be made to develop a radioimmunoassay. If developed, such an assay may be used for the early diagnosis of trophoblastic tumors and for following the course of treatment in patients with this disease. As an additional objective, we propose to examine the effect of proteolytic enzymes on the binding parameter of TSH receptors in thyroid membranes and to correlate it with any changes in adenylate cyclase activity. Our preliminary data suggest that the proposed studies may lead to an in vitro assay system with sensitivity for TSH.